Carboxypeptidase D Is an Avian Hepatitis B Virus Receptor

The receptor molecules for human and animal hepatitis B viruses have not been defined. Previous studies have described a 170 to 180 kDa molecule (p170 or gp180) that binds in vitro to the pre-S domain of the large envelope protein of duck hepatitis B virus (DHBV); cDNA cloning revealed the binding protein to be duck carboxypeptidase D (DCPD). In the present study, the DCPD cDNA was transfected into several nonpermissive human-, monkey-, and avian species-derived cell lines. Cells transfected with a plasmid encoding the full-length DCPD protein bound DHBV particles, whereas cells expressing truncated versions of DCPD protein that fail to bind the pre-S protein did not. The DHBV binding to DCPD-reconstituted cells was blocked by a monoclonal antibody that neutralizes DHBV infection of primary duck hepatocytes (PDH) and also by a pre-S peptide previously shown to inhibit DHBV infection of PDH. In addition to promoting virus binding, DCPD expression was associated with internalization of viral particles. The entry process was prevented by incubation of reconstituted cells with DHBV at 4 degrees C and by the addition of energy-depleting agents known to block DHBV entry into PDH. These results demonstrated that DCPD is a DHBV receptor. However, the lack of complete viral replication in DCPD-reconstituted cells suggested that additional factors are required for postentry events in immortalized cell lines.     

Superinfection exclusion in duck hepatitis B virus infection is mediated by the large surface antigen

Superinfection exclusion is the phenomenon whereby a virus prevents the subsequent infection of an already infected host cell. The Pekin duck hepatitis B virus (DHBV) model was used to investigate superinfection exclusion in hepadnavirus infections. Superinfection exclusion was shown to occur both in vivo and in vitro with a genetically marked DHBV, DHBV-ClaI, which was unable to establish an infection in either DHBV-infected ducklings or DHBV-infected primary duck hepatocytes (PDHs). In addition, exclusion occurred in vivo even when the second virus had a replicative advantage. Superinfection exclusion appears to be restricted to DHBV, as adenovirus, herpes simplex virus type 1, and vesicular stomatitis virus were all capable of efficiently infecting DHBV-infected PDHs. Exclusion was dependent on gene expression by the original infecting virus, since UV-irradiated DHBV was unable to mediate the exclusion of DHBV-ClaI. Using recombinant adenoviruses expressing DHBV proteins, we determined that the large surface antigen mediated exclusion. The large surface antigen is known to cause down-regulation of a DHBV receptor, carboxypeptidase D (CPD). Receptor down-regulation is a mechanism of superinfection exclusion seen in other viral infections, and so it was investigated as a possible mechanism of DHBV-mediated exclusion. However, a mutant large surface antigen which did not down-regulate CPD was still capable of inhibiting DHBV infection of PDHs. In addition, exclusion of DHBV-ClaI did not correlate with a decrease in CPD levels. Finally, virus binding assays and confocal microscopy analysis of infected PDHs indicated that the block in infection occurs after internalization of the second virus. We suggest that superinfection exclusion may result from the role of the L surface antigen as a regulator of intracellular trafficking.     

Carboxypeptidase D (gp180), a Golgi-Resident Protein, Functions in the Attachment and Entry of Avian Hepatitis B Viruses

Carboxypeptidase D (gp180), one of many candidate receptors proposed for hepatitis B viruses (HBVs), was examined and found to be the actual cellular receptor for avian HBVs. This conclusion was based on the following observations: (i) gp180 was the only host protein that bound with high affinity to the pre-S ectodomain of the large duck hepatitis B virus (DHBV) envelope protein, which is known to be essential for virus infection; (ii) a pre-S subdomain which determines physical binding to gp180 was found to coincide with a domain functionally defined in infection competition experiments as a receptor binding domain; (iii) soluble gp180, lacking the membrane anchor, efficiently inhibited DHBV infection; (iv) efficient interspecies gp180–pre-S interaction was limited to the natural hosts of avian hepadnaviruses; and (v) expression of gp180 in a heterologous hepatoma cell line mediated cellular attachment and subsequent internalization of fluorescently labeled viral particles into vesicular structures. However, gp180 expression did not render transfected heterologous cells permissive for productive infection, suggesting that a species-specific coreceptor is required for fusion to complete viral entry. In contrast to the case for known virus receptors, gp180 was not detected on the hepatocyte cell surface but was found to be concentrated in the Golgi apparatus, from where it functions by cycling to and from the plasma membrane.     

Envelope protein-mediated down-regulation of hepatitis B virus receptor in infected hepatocytes

Entry of duck hepatitis B virus (DHBV) is initiated by specific interaction of its large envelope protein (L) with a cellular entry receptor, recently identified as carboxypeptidase D (CPD; historically gp180). In this report, we present evidence demonstrating that this receptor is down-regulated as a result of DHBV infection: (i) receptor levels determined by Western blot were much reduced in DHBV-infected duck livers and undetectable by immunostaining in infected cultured hepatocytes; (ii) results from metabolic labeling experiments indicate enhanced receptor protein turnover; (iii) the kinetics of receptor loss from newly infected cells correlated with the accumulation of newly synthesized viral protein; (iv) expression of DHBV L protein, transduced from a recombinant adenovirus, was sufficient to eliminate gp180/CPD from the Golgi compartment, its normal predominant location; (v) gp180/CPD remained absent from the Golgi compartment in infected hepatocytes, even after overexpression from a recombinant adenovirus, while residual amounts subsequently became detectable in a perinuclear compartment, containing DHBV L protein; (vi) expression of DHBV L protein in a HepG2 cell line, stably expressing gp180/CPD, leads to incomplete receptor maturation and induces its degradation. Taken together, these data are consistent with a model in which the virus receptor interacts early in the biosynthetic pathway with the viral L protein, leading to its retention in a pre-Golgi compartment and to subsequent degradation, thus preventing receptor interference with the export of DHBV via the secretory pathway which it shares with its receptor. Accordingly, and analogously with receptor down-regulation in retroviral systems, DHBV receptor down-modulation may account for the much-reduced efficiency of DHBV superinfection of preinfected hepatocytes.     

Inhibition of Duck Hepatitis B Virus Infection by a Myristoylated Pre-S Peptide of the Large Viral Surface Protein

We have used the duck hepatitis B virus (DHBV) model to study the interference with infection by a myristoylated peptide representing an N-terminal pre-S subdomain of the large viral envelope protein. Although lacking the essential part of the carboxypeptidase D (formerly called gp180) receptor binding site, the peptide binds hepatocytes and subsequently blocks DHBV infection. Since its activity requires an amino acid sequence involved in host discrimination between DHBV and the related heron HBV (T. Ishikawa and D. Ganem, Proc. Natl. Acad. Sci. USA 92:6259-6263, 1995), we suggest that it is related to the postulated host-discriminating cofactor of infection.     

Avian Hepatitis B Virus Infection Is Initiated by the Interaction of a Distinct Pre-S Subdomain with the Cellular Receptor gp180

Functionally relevant hepadnavirus-cell surface interactions were investigated with the duck hepatitis B virus (DHBV) animal model by using an in vitro infection competition assay. Recombinant DHBV pre-S polypeptides, produced in Escherichia coli, were shown to inhibit DHBV infection in a dose-dependent manner, indicating that monomeric pre-S chains were capable of interfering with virus-receptor interaction. Particle-associated pre-S was, however, 30-fold more active, suggesting that cooperative interactions enhance particle binding. An 85-amino-acid pre-S sequence, spanning about half of the DHBV pre-S chain, was characterized by deletion analysis as essential for maximal inhibition. Pre-S polypeptides from heron hepatitis B virus (HHBV) competed DHBV infection equally well despite a 50% difference in amino acid sequence and a much-reduced infectivity of HHBV for duck hepatocytes. These observations are taken to indicate (i) that the functionality of the DHBV pre-S subdomain, which interacts with the cellular receptor, is determined predominantly by a defined three-dimensional structure rather than by primary sequence elements; (ii) that cellular uptake of hepadnaviruses is a multistep process involving more than a single cellular receptor component; and (iii) that gp180, a cellular receptor candidate unable to discriminate between DHBV and HHBV, is a common component of the cellular receptor complex for avian hepadnaviruses.     

Uptake of duck hepatitis B virus into hepatocytes occurs by endocytosis but does not require passage of the virus through an acidic intracellular compartment.

The infectious entry pathway of duck hepatitis B virus (DHBV) was investigated with primary duck hepatocytes. Virus uptake was measured by a selective PCR technique which allows for the detection of a successful infection without the need for viral replication or gene expression. To test whether DHBV uptake occurs by endocytosis, the effects of energy depletion were analyzed. The requirement for an acidic intracellular pH was tested with the lysosomotropic agent ammonium chloride. The data show that energy depletion prevents the uptake of DHBV into primary hepatocytes whereas ammonium chloride has no effect. From these data, we conclude that DHBV is taken up by its host cells by endocytosis. However, in contrast to that of most other enveloped viruses, escape of DHBV from the endocytotic route does not depend on an acidic intracellular compartment.     

Glycine decarboxylase mediates a postbinding step in duck hepatitis B virus infection

Envelope protein precursors of many viruses are processed by a basic endopeptidase to generate two molecules, one for receptor binding and the other for membrane fusion. Such a cleavage event has not been demonstrated for the hepatitis B virus family. Two binding partners for duck hepatitis B virus (DHBV) pre-S envelope protein have been identified. Duck carboxypeptidase D (DCPD) interacts with the full-length pre-S protein and is the DHBV docking receptor, while duck glycine decarboxylase (DGD) has the potential to bind several deletion constructs of the pre-S protein in vitro. Interestingly, DGD but not DCPD expression was diminished following prolonged culture of primary duck hepatocytes (PDH), which impaired productive DHBV infection. Introduction of exogenous DGD promoted formation of protein-free viral genome, suggesting restoration of several early events in viral life cycle. Conversely, blocking DGD expression in fresh PDH by antisense RNA abolished DHBV infection. Moreover, addition of DGD antibodies soon after virus binding reduced endogenous DGD protein levels and impaired production of covalently closed circular DNA, the template for DHBV gene expression and genome replication. Our findings implicate this second pre-S binding protein as a critical cellular factor for productive DHBV infection. We hypothesize that DCPD, a molecule cycling between the cell surface and the trans-Golgi network, targets DHBV particles to the secretary pathway for proteolytic cleavage of viral envelope protein. DGD represents the functional equivalent of other virus receptors in its interaction with processed viral particles.     

A cell surface protein that binds avian hepatitis B virus particles.

We have identified a 180-kDa cellular glycoprotein (gp180) that binds with high affinity to duck hepatitis B virus (DHBV) particles. The protein was detected by coprecipitating labeled duck hepatocyte proteins with virions or recombinant DHBV envelope proteins, using nonneutralizing monoclonal antibodies to the virion envelope. Binding of gp180 requires only the pre-S region of the viral large envelope protein, since recombinant fusion proteins bearing only this region efficiently coprecipitate gp180. The DHBV-gp180 interaction is blocked by two independent neutralizing monoclonal antibodies. The protein is found on both internal and surface membranes of the cell, and the species distribution of gp180 binding activity mirrors the known host range of DHBV infection. Functional gp180 is expressed in a wide variety of tissues in susceptible ducks.     

A hydrophobic domain in the large envelope protein is essential for fusion of duck hepatitis B virus at the late endosome

The duck hepatitis B virus (DHBV) envelope is comprised of two transmembrane (TM) proteins, the large (L) and the small (S), that assemble into virions and subviral particles. Secondary-structure predictions indicate that L and S have three alpha-helical, membrane-spanning domains, with TM1 predicted to act as the fusion peptide following endocytosis of DHBV into the hepatocyte. We used bafilomycin A1 during infection of primary duck hepatocytes to show that DHBV must be trafficked from the early to the late endosome for fusion to occur. Alanine substitution mutations in TM1 of L and S, which lowered TM1 hydrophobicity, were used to examine the role of TM1 in infectivity. The high hydrophobicity of the TM1 domain of L, but not of S, was shown to be essential for virus infection at a step downstream of receptor binding and virus internalization. Using wild-type and mutant synthetic peptides, we demonstrate that the hydrophobicity of this domain is required for the aggregation and the lipid mixing of phospholipid vesicles, supporting the role of TM1 as the fusion peptide. While lipid mixing occurred at pH 7, the kinetics of insertion of the fusion peptide was increased at pH 5, consistent with the location of DHBV in the late-endosome compartment and previous studies of the nonessential role of low pH for infectivity. Exchange of the TM1 of DHBV with that of hepatitis B virus yielded functional, infectious DHBV particles, suggesting that TM1 of all of the hepadnaviruses act similarly in the fusion mechanism.     

A short sequence within domain C of duck carboxypeptidase D is critical for duck hepatitis B virus binding and determines host specificity

Virus-cell surface receptor interactions are of major interest. Hepadnaviruses are a family of partially double-stranded DNA viruses with liver tropism and a narrow host range of susceptibility to infection. At least in the case of duck hepatitis B virus (DHBV), host specificity seems controlled partly at the receptor level. The middle portion in the pre-S region of the viral large envelope protein binds specifically to duck carboxypeptidase D (DCPD) but not to its human or chicken homologue. Although domain C of DCPD is implicated in ligand binding, the exact pre-S contact site remains to be determined. We prepared and tested a panel of chimeric constructs consisting of DCPD and human carboxypeptidase D (HCPD). Our results indicate that a short region at the N terminus of domain C (residues 920 to 949) is critical to DHBV binding and is a major determinant for the host specificity of DHBV infection. Replacing this region of the DCPD molecule with its human homologue abolished the DHBV interaction, whereas introducing this DCPD sequence into HCPD conferred efficient DHBV binding. Extensive analysis of site-directed mutants revealed that both conserved and nonconserved residues were important for the pre-S interaction. There were primary sequence variations and secondary structural differences that contributed to the inability of HCPD to bind the DHBV pre-S domain.     

A soluble form of the avian hepatitis B virus receptor. Biochemical characterization and functional analysis of the receptor ligand complex

Avian hepatitis B virus infection is initiated by the specific interaction of the extracellular preS part of the large viral envelope protein with carboxypeptidase D (gp180), the primary cellular receptor. To functionally and biochemically characterize this interaction, we purified a soluble form of duck carboxypeptidase D from a baculovirus expression system, confirmed its receptor function, and investigated the contribution of different preS sequence elements to receptor binding by surface plasmon resonance analysis. We found that preS binds duck carboxypeptidase D with a 1:1 stoichiometry, thereby inducing conformational changes but not oligomerization. The association constant of the complex was determined to be 2.2 x 10(7) M-1 at 37 degreesC, pH 7.4, with an association rate of 4.0 x 10(4) M-1 s-1 and a dissociation rate of 1.9 x 10(-3) s-1, substantiating high affinity interaction of avihepadnaviruses with their receptor carboxypeptidase D. The separately expressed receptor-binding domain, comprising about 50% of preS as defined by mutational analysis, exhibits similar constants. The domain consists of an essential element, probably responsible for the initial receptor contact and a part that contributes to complex stabilization in a conformation sensitive manner. Together with previous results from cell biological studies these data provide new insights into the initial step of hepadnaviral infection.     

The earliest steps in hepatitis B virus infection

The early steps in hepatitis B virus (HBV) infection, a human hepadnavirus, initiates from cell attachment followed by entry and delivery of the genetic information to the nucleus. Despite the fact that these steps determine the virus-related pathogenesis, their molecular basis is poorly understood. Cumulative data suggest that this process can be divided to cell attachment, endocytosis, membrane fusion and post-fusion consecutive steps. These steps are likely to be regulated by the viral envelope proteins and by the cellular membrane, receptors and extracellular matrix. In the absence of animal model for HBV, the duck hepadnavirus DHBV turned out to be a fruitful animal model. Therefore data concerning the early, post-attachment steps in hepadnaviral entry are largely based on studies performed with DHBV in primary duck liver hepatocytes. These studies are now starting to illuminate the mechanisms of hepadnavirus route of cell entry and to provide some new insights on the molecular basis of the strict species specificity of hepadnavirus infection.     

The earliest steps in hepatitis B virus infection

The early steps in hepatitis B virus (HBV) infection, a human hepadnavirus, initiates from cell attachment followed by entry and delivery of the genetic information to the nucleus. Despite the fact that these steps determine the virus-related pathogenesis, their molecular basis is poorly understood. Cumulative data suggest that this process can be divided to cell attachment, endocytosis, membrane fusion and post-fusion consecutive steps. These steps are likely to be regulated by the viral envelope proteins and by the cellular membrane, receptors and extracellular matrix. In the absence of animal model for HBV, the duck hepadnavirus DHBV turned out to be a fruitful animal model. Therefore data concerning the early, post-attachment steps in hepadnaviral entry are largely based on studies performed with DHBV in primary duck liver hepatocytes. These studies are now starting to illuminate the mechanisms of hepadnavirus route of cell entry and to provide some new insights on the molecular basis of the strict species specificity of hepadnavirus infection.     

Receptor recognition by a hepatitis B virus reveals a novel mode of high affinity virus-receptor interaction

The duck hepatitis B virus model system was used to elucidate the characteristics of receptor (carboxypeptidase D, gp180) interaction with polypeptides representing the receptor binding site in the preS part of the large viral surface protein. We demonstrate the pivotal role of carboxypeptidase D for virus entry and show its C-domain represents the virus attachment site, which binds preS with extraordinary affinity. Combining results from surface plasmon resonance spectroscopy and two-dimensional NMR analysis we resolved the contribution of preS sequence elements to complex stability and show that receptor binding potentially occurs in two steps. Initially, a short alpha-helix in the C-terminus of the receptor binding domain facilitates formation of a primary complex. This complex is stabilized sequentially, involving approximately 60 most randomly structured amino acids preceding the helix. Thus, hepadnaviruses exhibit a novel mechanism of high affinity receptor interaction by conserving the potential to adapt structure during binding rather than to preserve it per se. We propose that this process represents an alternative strategy to escape immune surveillance and the evolutionary pressure inherent in the compact hepadnaviral genome organization.     

Entry of duck hepatitis B virus into primary duck liver and kidney cells after discovery of a fusogenic region within the large surface protein

Hepatitis B viruses exhibit a narrow host range specificity that is believed to be mediated by a domain of the large surface protein, designated L. For duck hepatitis B virus, it has been shown that the pre-S domain of L binds to carboxypeptidase D, a cellular receptor present in many species on a wide variety of cell types. Nonetheless, only hepatocytes become infected. It has remained vague which viral features determine host range specificity and organotropicity. By using chymotrypsin to treat duck hepatitis B virus, we addressed the question of whether a putative fusogenic region within the amino-terminal end of the small surface protein may participate in viral entry and possibly constitute one of the determinants of the host range of the virus. Addition of the enzyme to virions resulted in increased infectivity. Remarkably, even remnants of enzyme-treated subviral particles proved to be inhibitory to infection. A noninfectious deletion mutant devoid of the binding region for carboxypeptidase D could be rendered infectious for primary duck hepatocytes by treatment with chymotrypsin. Although because of the protease treatment mutant and wild-type viruses may have become infectious in an unspecific and receptor-independent manner, their host range specificity was not affected, as shown by the inability of the virus to replicate in different hepatoma cell lines, as well as primary chicken hepatocytes. Instead, the organotropicity of the virus could be reduced, which was demonstrated by infection of primary duck kidney cells.     

Characterization of a 120-Kilodalton pre-S-binding protein as a candidate duck hepatitis B virus receptor.

Infection by human and animal hepadnaviruses displays remarkable host and tissue tropism. The infection cycle probably initiates with binding of the pre-S domain of viral envelope protein to surface receptors present on the hepatocyte. Three types of neutralizing monoclonal antibodies against duck hepatitis B virus (DHBV) have their binding sites clustered within residues 83 to 107 of the pre-S protein, suggesting that this region may constitute a major receptor binding site. A 170- or 180-kDa duck protein (p170 or gp180) which binds DHBV particles through this part of the pre-S sequence has been identified recently. Although the p170 binding protein is host (duck) specific, its distribution is not restricted to DHBV-infectible tissues. Using the pre-S protein fused to glutathione S-transferase and immobilized on Sepharose beads, we have now identified an additional binding protein with a size of 120 kDa (p120). p120 expression is restricted to the liver, kidney, and pancreas, the three major organs of DHBV replication. While optimal p170 binding requires an intact pre-S protein, binding to p120 occurs much more efficiently with a few N- or C-terminally truncated forms. The p120 binding site was mapped to residues 98 to 102 of the pre-S region, which overlaps with a cluster of known virus-neutralizing epitopes. Site-directed mutagenesis revealed residues 100 to 102 (Phe-Arg-Arg) as the critical p120 contact site; nonconservative substitution in any of the three positions abolished p120 binding. Double mutations at positions 100 to 102 markedly reduced DHBV infectivity in cell culture. Short pre-S peptides covering the clustered neutralizing epitopes (also p170 and p120 binding sites) reduced DHBV infectivity in primary duck hepatocyte cultures. Thus, p120 represents a candidate component of the DHBV receptor complex.     

氧化苦参碱在鸭原代肝细胞中抗鸭乙肝病毒(DHBV)作用的研究

通过建立鸭原代肝细胞-DHBV感染模型研究氧化苦参碱抗DHBV的作用。分别在DHBV感染前、感染同时以及感染后给药,利用打点杂交、Southern印迹核酸杂交和荧光定量PCR方法分别检测培养细胞上清及细胞内病毒核酸,观察氧化苦参碱在病毒感染的各个环节所起的抗病毒作用。实验结果显示:1mg/mL氧化苦参碱处理细胞后,鸭原代肝细胞培养上清及细胞内的DHBV核酸明显低于病毒感染对照组,病毒抑制率达91.6%;在病毒感染同时加药对病毒的抑制率可达98.5%;感染后持续用药能使不同培养天数的鸭肝细胞内的DHBV核酸降低60.5%~96.6%;氧化苦参碱与DHBV共孵育后,可以使病毒感染力下降69.6%。结果说明氧化苦参碱可以在DHBV感染鸭原代肝细胞的多个环节,包括病毒吸附、进入细胞及细胞内复制等方面发挥抗病毒作用。     

New hepatitis B virus of cranes that has an unexpected broad host range

All hepadnaviruses known so far have a very limited host range, restricted to their natural hosts and a few closely related species. This is thought to be due mainly to sequence divergence in the large envelope protein and species-specific differences in host components essential for virus propagation. Here we report an infection of cranes with a novel hepadnavirus, designated CHBV, that has an unexpectedly broad host range and is only distantly evolutionarily related to avihepadnaviruses of related hosts. Direct DNA sequencing of amplified CHBV DNA as well a sequencing of cloned viral genomes revealed that CHBV is most closely related to, although distinct from, Ross' goose hepatitis B virus (RGHBV) and slightly less closely related to duck hepatitis B virus (DHBV). Phylogenetically, cranes are very distant from geese and ducks and are most closely related to herons and storks. Naturally occurring hepadnaviruses in the last two species are highly divergent in sequence from RGHBV and DHBV and do not infect ducks or do so only marginally. In contrast, CHBV from crane sera and recombinant CHBV produced from LMH cells infected primary duck hepatocytes almost as efficiently as DHBV did. This is the first report of a rather broad host range of an avihepadnavirus. Our data imply either usage of similar or identical entry pathways and receptors by DHBV and CHBV, unusual host and virus adaptation mechanisms, or divergent evolution of the host genomes and cellular components required for virus propagation.